Methods of using and compositions comprising immunomodulatory compounds for the treatment and management of myelodysplastic syndromes

ABSTRACT

Methods of treating, preventing and/or managing myclodysplastic syndromes are disclosed. Specific methods encompass the administration of an immunomodulatory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, alone or in combination with a second active ingredient, and/or the transplantation of blood or cells. Specific second active ingredients are capable of affecting or blood cell production. Pharmaceutical compositions, single unit dosage forms, and kits suitable for use in methods of the invention are also disclosed.

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/418,468 filed on Oct. 15, 2002, the entirety of whichis incorporated herein by reference.

1. FIELD OF THE INVENTION

[0002] This invention relates to methods of treating, preventing and/ormanaging myelodysplastic and related syndromes which comprise theadministration of immunomodulatory compounds alone or in combinationwith known therapeutics. The invention also relates to pharmaceuticalcompositions and dosing regimens. In particular, the inventionencompasses the use of immunomodulatory compounds in conjunction withtransplantation therapy and/or other standard therapies formyelodysplastic syndromes.

2. BACKGROUND OF THE INVENTION

[0003] 2.1. Pathobiology of MDS

[0004] Myelodysplastic syndrome (“MDS”) refers to a diverse group ofhematopoietic stem cell disorders. MDS is characterized by a cellularmarrow with impaired morphology and maturation (dysmyelopoiesis),peripheral blood cytopenias, and a variable risk of progression to acuteleukemia, resulting from ineffective blood cell production. The MerckManual 953 (17^(th) ed. 1999) and List et al., 1990, J Clin. Oncol.8:1424.

[0005] The initial hematopoictic stem cell injury can be from causessuch as, but not limited to, cytotoxic chemotherapy, radiation, virus,chemical exposure, and genetic predisposition. A clonal mutationpredominates over bone marrow, suppressing healthy stem cells. In theearly stages of MDS, the main cause of cytopenias is increasedprogrammed cell death (apoptosis). As the disease progresses andconverts into leukemia, gene mutation rarely occurs and a proliferationof leukemic cells overwhelms the healthy marrow. The disease coursediffers, with some cases behaving as an indolent disease and othersbehaving aggressively with a very short clinical course that convertsinto an acute form of leukemia.

[0006] The actual incidence of MDS in the U.S. is unknown. MDS was firstconsidered a distinct disease in 1976, and occurrence was estimated at1500 new cases every year. At that time, only patients with less thanfive percent blasts were considered to have this disorder. Statisticsfrom 1999 estimated 13,000 new cases per year and about 1000 cases peryear in children, surpassing chronic lymphocytic leukemia as the mostcommon form of leukemia in the western hemisphere. The perception thatthe incidence is increasing may be due to improvements in recognitionand criteria for diagnosis. The disease is found worldwide.

[0007] An international group of hematologists, theFrench-American-British (FAB) Cooperative Group, classified MDSdisorders into five subgroups, differentiating them from acute myeloidleukemia. The Merck Manual 954 (17^(th) ed. 1999); Bennett J. M., etal., Ann. Intern. Med. 1985 October, 103(4): 620-5; and Besa E. C., Med.Clin. North Am. 1992 May, 76(3): 599-617. An underlying trilineagedysplastic change in the bone marrow cells of the patients is found inall subtypes.

[0008] There are two subgroups of refractory anemia characterized byfive percent or less myeloblasts in bone marrow: (1) refractory anemia(RA) and; (2) RA with ringed sideroblasts (RARS), definedmorphologically as having 15% erythroid cells with abnormal ringedsideroblasts, reflecting an abnormal iron accumulation in themitochondria. Both have a prolonged clinical course and low incidence ofprogression to acute leukemia. Besa E. C., Med. Clin. North Am. 1992May, 76(3): 599-617.

[0009] There are two subgroups of refractory anemias with greater thanfive percent mycloblasts: (1) RA with excess blasts (RAEB), defined as6-20% myeloblasts, and (2) RAEB in transformation (RAEB-T), with 21-30%myeloblasts. The higher the percentage of myeloblasts, the shorter theclinical course and the closer the disease is to acute myelogenousleukemia. Patient transition from early to more advanced stagesindicates that these subtypes are merely stages of disease rather thandistinct entities. Elderly patients with MDS with trilineage dysplasiaand greater than 30% myeloblasts who progress to acute leukemia areoften considered to have a poor prognosis because their response rate tochemotherapy is lower than de novo acute myeloid leukemia patients. TheWorld Health Organization (WHO) classification (1999) proposes toinclude all cases of RAEB-T, or patients with greater than 20%myeloblasts, in the category of acute leukemia because these patientshave similar prognostic outcomes. However, their response to therapy isworse than the de novo or more typical acute myelogenous leukemia oracute nonlymphocytic leukemia (ANLL) patient. Id.

[0010] The fifth type of MDS, the most difficult to classify, is calledchronic myclomonocytic leukemia (CMML). This subtype can have anypercentage of myeloblasts but presents with a monocytosis of 1000/dL ormore. It may be associated with splenomegaly. This subtype overlaps witha myeloproliferative disorder and may have an intermediate clinicalcourse. It is differentiated from the classic chronic myelocyticleukemia (CML) that is characterized by a negative Ph chromosome. Therecent WHO classification (1999) proposes that juvenile andproliferative CMML be listed separately from FAB underMDS/myeloproliferative disorder (MPD) with splenomegaly and greater than13,000 total WBC. CMML is limited to monocytosis, less than 13,000/mm³total leukocytes, and requires trilineage dysplasia. Id. Harris N. L.,et al., J. Clin. Oncol. 1999 December, 17(12): 3835-49. Finally, someother international organizations, including WHO, have suggested a sixthclass of MDS patients, characterized by a del (5q) abnormality.

[0011] MDS is primarily a disease of elderly people, with the medianonset in the seventh decade of life. The median age of these patients is65 years, with ages ranging from the early third decade of life to asold as 80 years or older. The syndrome may occur in any age group,including the pediatric population. Patients who survive malignancytreatment with alkylating agents, with or without radiotherapy, have ahigh incidence of developing MDS or secondary acute leukemia. About60-70% of patients do not have an obvious exposure or cause for MDS, andare classified as primary MDS patients.

[0012] The most common cases of MDS are primary, or idiopathic. However,a nonspecific history of exposure to indeterminable chemicals orradiation 10-15 years prior to onset of disease may be present in about50% of patients. This relationship to pathogenesis remains unproved.Compounds such as, but not limited to, benzene, insecticides, weedkillers, and fungicides are possible causes of MDS. Goldberg H., et al.,Cancer Res. 1990 Nov. 1; 50(21): 6876-81. Secondary MDS describesdevelopment of MDS or acute leukemia after known exposures tochemotherapy drugs that can cause bone marrow damage. These drugs areassociated with a high incidence of chromosomal abnormalities followingexposure and at the time of MDS or acute leukemia diagnosis.

[0013] Further, MDS is associated with complications associated withsevere cytopenias. Other complications are development of myelofibrosis,which can accelerate decline in blood counts and increase transfusionrequirements. Transformation to acute leukemia accelerates thedevelopment of complications such as anemia, bleeding, and infections.

[0014] Recently, the International MDS Risk Analysis (IMRA) Workshopproposed an International Prognosis Scoring System (IPSS) to decreaseimprecision in predicting survival and AML risk in MDS patients. TheIPSS is based on the number of cytopenias, percentage of BM blasts, andtype of cytogenetic abnormalities (Table 1). Greenberg et al., Blood1997, 89:2079-88. The latter are categorized into good (normal, -Y, del(5q), del (20q)), intermediate, and poor subgroups (complex orchromosome 7 abnormalities). TABLE 1 International Prognostic ScoringSystem for MDS Score Value Prognostic 0 0.5 1.0 1.5 2.0 VariableBonemarrow <5 5-10 — 11-20 21-30 blasts (%) Karyotype* Good IntermediatePoor Cytopenias 0/1 2/3

[0015] 2.2. MDS Treatment

[0016] The current treatment of MDS is based on the stage and themechanism of the disease that predominates the particular phase of thedisease process. Bone marrow transplantation has been used in patientswith poor prognosis or late-stage MDS. Epstein and Slease, 1985, Surg.Ann. 17:125. This type of therapy, however, is both painful for donorand recipient, because of the involvement of invasive procedures and cancause severe and even fatal complications to the recipient, particularlywith allogeneic transplant and related Graft Versus Host Disease (GVHD)results. Therefore, the risk of GVHD restricts the use of bone marrowtransplantation to patients with otherwise fatal diseases. Further, asmost patients are elderly and only a few young MDS patients will have amatched donor, the use of bone marrow transplantation is limited.

[0017] An alternative approach to therapy for MDS is the use ofhematopoietic growth factors or cytokines to stimulate blood celldevelopment in a recipient. Dexter, 1987, J. Cell Sci. 88:1; Moore,1991, Annu. Rev. Immunol. 9:159; and Besa E. C., Med. Clin. North Am.1992 May, 76(3): 599-617. The process of blood cell formation, by whicha small number of self-renewing stem cells give rise to lineage specificprogenitor cells that subsequently undergo proliferation anddifferentiation to produce the mature circulating blood cells has beenshown to be at least in part regulated by specific hormones. Thesehormones are collectively known as hematopoietic growth factors.Metcalf, 1985, Science 229:16; Dexter, 1987,J. Cell Sci. 88:1; Golde andGasson, 1988, Scientific American, July:62; Tabbara and Robinson, 1991,Anti-Cancer Res. 11:81; Ogawa, 1989, Environ. Health Presp. 80:199; andDexter, 1989, Br. Med. Bull. 45:337. The most well characterized growthfactors include erythropoietin (EPO), granulocyte macrophage colonystimulating factor (GM-CSF), and granulocyte colony stimulating factor(G-CSF). Apart from inducing proliferation and differentiation ofhematopoietic progenitor cells, such cytokines have also been shown toactivate a number of functions of mature blood cells, includinginfluencing the migration of mature hematopoietic cells. Stanley et al.,1976, J. Exp. Med. 143:631; Schrader et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:323; Moore et al., 1980, J. Immunol. 125:1302; Kurland etal., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:2326; Handman and Burgess,1979, J. Immunol. 122:1134; Vadas et al., 1983, Blood 61:1232; Vadas etal., 1983, J. Immunol. 130:795; and Weibart et al., 1986, J. Immunol.137:3584.

[0018] Unfortunately, hematopoietic growth factors have not proveneffective in many clinical settings. Clinical trials of MDS patientstreated with recombinant human GM-CSF and G-CSF have shown that whilethese cytokines can restore granulocytopoiesis in treated patients,their efficacy is restricted to the granulocyte or monocyte lineage withlittle or no improvement in hemoglobin or platelet counts. Schuster etal., 1990, Blood 76 (Suppl.1):318a. When such patients were treated withrecombinant human EPO, a sustained improvement in hemoglobin or decreasein transfusion requirement was achieved in only less than 25% ofpatients. Besa et al., 1990, 76 (Suppl.1):133a; Hellstrom et al., 1990,76 (Suppl.1):279a; Bowen et al., 1991, Br. J. Haematol. 77:419.Therefore, there remains a need for safe and effective methods oftreating and managing MDS.

[0019] 2.3. Thalidomide and Other Compounds Useful in the Treatment ofDisease

[0020] Thalidomide is a racemic compound sold under the tradenameThalomid® and chemically named α-(N-phthalimido)glutarimide or2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione. Thalidomide wasoriginally developed in the 1950's to treat morning sickness, but due toits teratogenic effects was withdrawn from use. Thalidomide has beenapproved in the United States for the acute treatment of the cutaneousmanifestations of erythema nodosum leprosum in leprosy. Physicians' DeskReference, 25 1154-1158 (56th ed., 2002). Because its administration topregnant women can cause birth defects, the sale of thalidomide isstrictly controlled. Id. Thalidomide has reportedly been studied in thetreatment of other diseases, such as chronic graft-vs-host disease,rheumatoid arthritis, sarcoidosis, several inflammatory skin diseases,and inflammatory bowel disease. See generally, Koch, H. P., Prog. Med.Chem. 22:165-242 (1985). See also, Moller, D. R., et al., J. Immunol.159:5157-5161 (1997); Vasiliauskas, E. A., et al., Gastroenterology117:1278-1287 (1999); Ehrenpreis, E. D., et al., Gastroenterology117:1271-1277 (1999). It has further been alleged that thalidomide canbe combined with other drugs to treat ischemia/repercussion associatedwith coronary and cerebral occlusion. See U.S. Pat. No. 5,643,915, whichis incorporated herein by reference.

[0021] More recently, thalidomide was found to exert immunomodulatoryand anti-inflammatory effects in a variety of disease states, cachexiain AIDS, and opportunic infections in AIDS. In studies to define thephysiological targets of thalidomide, the drug was found to have a widevariety of biological activities exclusive of its sedative effectincluding neurotoxicity, teratogenicity, suppression of TNF-α productionby monocytes/macrophages and the accompanying inflammatory toxicitiesassociated with high levels of TNF-α, and inhibition of angiogenesis andneovascularization.

[0022] Additionally, beneficial effects have been observed in a varietyof dermatological conditions, ulcerative colitis, Crohn's disease,Bechets's syndrome, systemic lupus erythematosis, aphthous ulcers, andlupus. The anti-angiogenic properties of thalidomide in in vivo modelshave been reported. D'Amato et al., Thalidomide Is An Inhibitor OfAngiogenesis, 1994, PNAS, USA 91:4082-4085.

[0023] One of the most therapeutically significant potential uses ofthalidomide is in the treatment of cancer. The compound has beeninvestigated in the treatment of various types of cancer, such asrefractory multiple myeloma, brain, breast, colon, and prostate cancer,melanoma, mesothelioma, and renal cell carcinoma. See, e.g., Singhal,S., et al., New England J. Med. 341(21):1565-1571 (1999); and Marx, G.M., et al., Proc. Am. Soc. Clin. Oncology 18:454a (1999). Thalidomidereportedly can also be used to prevent the development of chroniccardiomyopathy in rats caused by doxorubicin. Costa, P. T., et al.,Blood 92(10:suppl. 1):235b (1998). Other reports concerning the use ofthalidomide in the treatment of specific cancers include its combinationwith carboplatin in the treatment of glioblastoma multiforme. McCann,J., Drug Topics 41-42 (Jun. 21, 1999). The use of thalidomide incombination with dexamethasone reportedly was effective in the treatmentof patients suffering from multiple myeloma who also received, assupportive care, human granulocyte colony-stimulating factor (G-CSF),ciprofloxacin, and non-absorbable antifungal agents. Kropff, M. H.,Blood 96(11 part 1):168a (2000); see also, Munshi, N. et al., Blood94(10 part 1):578a (1999). Other chemotherapy combinations that comprisethalidomide are disclosed in International Application No.PCT/US01/15326 to R. Govindarjan and A. Zeitlan, and in InternationalApplication No. PCT/USO1/15327 to J. B. Zeldis, et al.

[0024] In an effort to provide compounds that have greater therapeuticsafety and efficacy than thalidomide, researchers have beguninvestigating a large number of other compounds, some of which arederivatives of thalidomide. See, e.g., Marriott, J. B., et al., ExpertOpin. Biol. Ther. 1(4):1-8 (2001); G. W. Muller, et al., Journal ofMedicinal Chemistry 39(17): 3238-3240 (1996); and G. W. Muller, et al.,Bioorganic & Medicinal Chemistry Letters 8: 2669-2674 (1998). Examplesinclude, but are not limited to, the substituted2-(2,6-dioxopiperidin-3-yl) phthalimies and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles described in U.S. Pat. Nos.6,281,230 and 6,316,471, both to G. W. Muller, et al.

[0025] A group of compounds selected for their capacity to potentlyinhibit TNF-α production by LPS stimulated PBMC has been investigated.L. G. Corral, et al., Ann. Rheum. Dis. 58:(Suppl I) 1107-1113 (1999).These compounds, which are referred to as IMiDs™ or ImmunomodulatoryDrugs, show not only potent inhibition of TNF-α but also markedinhibition of LPS induced monocyte IL1β and IL12 production. LPS inducedIL6 is also inhibited by IMiDs™, albeit partially. These compounds arepotent stimulators of LPS induced IL10, increasing IL10 levels by 200 to300%. Id.

[0026] While many such compounds have shown promise as therapeuticagents, their mechanisms of action and effectiveness are still underinvestigation. Moreover, there remains a need for therapeutic agents totreat MDS and its related disorders.

3. SUMMARY OF THE INVENTION

[0027] This invention encompasses methods of treating or preventingmyelodysplastic syndrome (“MDS”) which comprise administering to apatient in need thereof a therapeutically or prophylactically effectiveamount of an immunomodulatory compound of the invention or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof. The invention also encompasses methods ofmanaging MDS (e.g., lengthening the time of remission) which compriseadministering to a patient in need of such management a therapeuticallyor prophylactically effective amount of an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof.

[0028] One embodiment of the invention encompasses the use of one ormore immunomodulatory compounds in combination with conventionaltherapies presently used to treat, prevent or manage MDS such ashematopoietic growth factors, cytokines, cancer chemotherapeutics, stemcell transplantation and other transplantations.

[0029] The invention further encompasses pharmaceutical compositions,single unit dosage forms, and kits suitable for use in treating,preventing and/or managing MDS, which comprise an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof.

4. DETAILED DESCRIPTION OF THE INVENTION

[0030] A first embodiment of the invention encompasses methods oftreating or preventing MDS which comprise administering to a patient inneed of such treatment or prevention a therapeutically orprophylactically effective amount of an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof. The embodiment encompasses the treatment,prevention or management of specific sub-types of MDS such as refractoryanemia, refractory anemia with ringed sideroblasts, refractory anemiawith excess blasts, refractory anemia with excess blasts intransformation and chronic myelomonocytic leukemia.

[0031] As used herein, the term “myelodysplastic syndromes” or “MDS”means hematopoietic stem cell disorders characterized by one or more ofthe following: ineffective blood cell production, progressivecytopenias, risk of progression to acute leukemia or cellular marrowwith impaired morphology and maturation (dysmyelopoiesis). The term“myelodysplastic syndromes” or “MDS” unless otherwise noted includes:refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation and chronic myelomonocytic leukemia.

[0032] Another embodiment of the invention encompasses methods ofmanaging MDS which comprises administering to a patient in need of suchmanagement a prophylactically effective amount of an immunomodulatorycompound, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof.

[0033] Another embodiment of the invention encompasses a pharmaceuticalcomposition comprising an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

[0034] Also encompassed by the invention are single unit dosage formscomprising an immunomodulatory compound, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof.

[0035] Another embodiment of the invention encompasses a kit comprising:a pharmaceutical composition comprising an immunomodulatory compound, ora pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof and a second active or dexamethasone orinstructions for use. The invention further encompasses kits comprisingsingle unit dosage forms.

[0036] One embodiment of the invention encompasses a method of treating,preventing and/or managing MDS, which comprises administering to apatient in need of such treatment, prevention and/or management atherapeutically or prophylactically effective amount of animmunomodulatory compound, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and atherapeutically or prophylactically effective amount of a second activeagent.

[0037] The second active agent is preferably a hematopoietic growthfactor, a cytokine, an anti-cancer agent, an antibiotic, an anti-fungal,an anti-inflammatory, an immunosuppressive agent such as a cyclosporin,conventional therapy for MDS, or other chemotherapeutic agent found forexample in the Physician's Desk Reference 2002. Preferred anti-cancer orcancer chemotherapeutics are apoptosis inducing agents, topoisomeraseinhibitors, anti-angiogenesis compounds, microtubule stabilizing agents,alkylating agents and other known conventional cancer chemotherapy. Mostpreferred second active agents are those capable of affecting orimproving blood production. Second active agents can be large molecules(e.g., proteins) or small molecules (e.g., synthetic inorganic,organometallic, or organic molecules). The examples of specific secondactive agent include, but are not limited to, etanercept (Enbrel®),imatinib (Glivec®), anti-TNF-α antibodies, infliximab (Remicade®),G-CSF, GM-CSF, EPO, topotecan, irinotecan, pentoxifylline,ciprofloxacin, dexamethasone, IL2, IL8, IL18, Ara-C, vinorelbine,vinblastine, isotretinoin, and13-cis-retinoic acid. This invention alsoencompasses the use of native, naturally occurring, and recombinantproteins. The invention further encompasses mutants and derivatives(e.g., modified forms) of naturally occurring proteins that exhibit, invivo, at least some of the pharmacological activity of the proteins uponwhich they are based. Examples of mutants include, but are not limitedto, proteins that have one or more amino acid residues that differ fromthe corresponding residues in the naturally occurring forms of theproteins. Also encompassed by the term “mutants” are proteins that lackcarbohydrate moieties normally present in their naturally occurringforms (e.g., nonglycosylated forms). Examples of derivatives include,but are not limited to, pegylated derivatives and fusion proteins, suchas proteins formed by fusing IgG1 or IgG3 to the protein or activeportion of the protein of interest. See, e.g., Penichet, M. L. andMorrison, S. L., J. Immunol. Methods 248:91-101 (2001). Vaccines thatcause the secretion of proteins disclosed herein as well aspharmacologically active mutants, derivatives, and fusion thereof arealso encompassed by the invention.

[0038] Without being limited by theory, it is believed that certainimmunomodulatory compounds and proteins can act in complementary orsynergistic ways in the treatment or management of MDS. It is alsobelieved that certain proteins may reduce or eliminate particularadverse effects associated with some immunomodulatory compounds, therebyallowing the administration of larger amounts of an immunomodulatorycompound to patients and/or increasing patient compliance. It is furtherbelieved that some immunomodulatory compounds may reduce or eliminateparticular adverse effects associated with some protein-based MDStherapies, thereby allowing the administration of larger amounts ofprotein to patients and/or increasing patient compliance.

[0039] Another embodiment of the invention encompasses a method ofreversing, reducing or avoiding an adverse effect associated with theadministration of a chemotherapeutics or therapeutics used to treatcancer or MDS in a patient suffering from MDS, which comprisesadministering to a patient in need thereof a therapeutically orprophylactically effective amount of an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

[0040] As inevitable leukemic transformation develops in certain stagesof MDS, transplantation of peripheral blood stem cells, hematopoieticstem cell preparation or bone marrow may be necessary. It is believedthat the combined use of an immunomodulatory compound andtransplantation of stem cells in a patient suffering from MDS provides aunique and unexpected synergism. In particular, without being limited bytheory, it is believed that an immunomodulatory compound exhibitsimmunomodulatory activity that may provide additive or synergisticeffects when given concurrently with transplantation therapy.Immunomodulatory compounds can work in combination with transplantationtherapy reducing complications associated with the invasive procedure oftransplantation and risk of related Graft Versus Host Disease (GVHD).Therefore, this invention encompasses a method of treating, preventingand/or managing MDS, which comprises administering to a patient (e.g., ahuman) an immunomodulatory compound, or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof,before, during, or after transplantation therapy.

[0041] The invention also encompasses pharmaceutical compositions,single unit dosage forms, and kits which comprise one or moreimmunomodulatory compounds, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, a secondactive ingredient, and/or blood or cells for transplantation therapy.For example, the kit may contain one or more compounds of the invention,stem cells for transplantation and an immunosuppressive agent,antibiotic or other drug, each of which is to be used to treat the MDSpatient.

[0042] 4.1. Immunomodulatory Compounds

[0043] Compounds used in the invention include immunomodulatorycompounds that are racemic, stereomerically enriched or stereomericallypure, and pharmaceutically acceptable salts, solvates, hydrates,stereoisomers, clathrates, and prodrugs thereof. Preferred compoundsused in the invention are small organic molecules having a molecularweight less than about 1000 g/mol, and are not proteins, peptides,oligonucleotides, oligosaccharides or other macromolecules.

[0044] As used herein and unless otherwise indicated, the term“stereomerically pure” means a composition that comprises onestereoisomer of a compound and is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecomposition of a compound having one chiral center will be substantiallyfree of the opposite enantiomer of the compound. A stereomerically purecomposition of a compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, more preferably greater than about90% by weight of one stereoisomer of the compound and less than about10% by weight of the other stereoisomers of the compound, even morepreferably greater than about 95% by weight of one stereoisomer of thecompound and less than about 5% by weight of the other stereoisomers ofthe compound, and most preferably greater than about 97% by weight ofone stereoisomer of the compound and less than about 3% by weight of theother stereoisomers of the compound. As used herein and unless otherwiseindicated, the term “stereomerically enriched” means a composition thatcomprises greater than about 60% by weight of one stereoisomer of acompound, preferably greater than about 70% by weight, more preferablygreater than about 80% by weight of one stereoisomer of a compound. Asused herein and unless otherwise indicated, the term “enantiomericallypure” means a stereomerically pure composition of a compound having onechiral center. Similarly, the term “stereomerically enriched” means astereomerically enriched composition of a compound having one chiralcenter.

[0045] As used herein and unless otherwise indicated, the term“immunomodulatory compounds” or “IMiDs™” (Celgene Corporation) usedherein encompasses small organic molecules that markedly inhibit TNF-α,LPS induced monocyte IL1β and IL12, and partially inhibit IL6production. Specific immunomodulatory compounds of the invention arediscussed below.

[0046] TNF-α is an inflammatory cytokine produced by macrophages andmonocytes during acute inflammation. TNF-α is responsible for a diverserange of signaling events within cells. TNF-α may play a pathologicalrole in cancer. Without being limited by particular theory, one of thebiological effects exerted by the immunomodulatory compounds of theinvention is the reduction of synthesis of TNF-α. Immunomodulatorycompounds of the invention enhance the degradation of TNF-α mRNA.

[0047] Further, without being limited by particular theory,immunomodulatory compounds used in the invention may also be potentco-stimulators of T cells and increase cell proliferation dramaticallyin a dose dependent manner. Immunomodulatory compounds of the inventionmay also have a greater co-stimulatory effect on the CD8+ T cell subsetthan on the CD4+ T cell subset. In addition, the compounds preferablyhave anti-inflammatory properties, and efficiently co-stimulate T cells.

[0048] Specific examples of immunomodulatory compounds of the invention,include, but are not limited to, cyano and carboxy derivatives ofsubstituted styrenes such as those disclosed in U.S. Pat. No. 5,929,117;1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such asthose described in U.S. Pat. No. 5,874,448; the tetra substituted2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines(e.g., 4-methyl derivatives of thalidomide and EM-12), including, butnot limited to, those disclosed in U.S. Pat. No. 5,635,517; and a classof non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579and 5,877,200; analogs and derivatives of thalidomide, includinghydrolysis products, metabolites, derivatives and precursors ofthalidomide, such as those described in U.S. Pat. Nos. 5,593,990,5,629,327, and 6,071,948 to D'Amato; aminothalidomide, as well asanalogs, hydrolysis products, metabolites, derivatives and precursors ofaminothalidomide, and substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolessuch as those described in U.S. Pat. Nos. 6,281,230 and 6,316,471;isoindole-imide compounds such as those described in U.S. patentapplication Ser. No. 09/972,487 filed on Oct. 5, 2001, U.S. patentapplication Ser. No.10/032,286 filed on Dec. 21, 2001, and InternationalApplication No. PCT/US01/50401 (International Publication No. WO02/059106). The entireties of each of the patents identified herein areincorporated herein by reference. Immunomodulatory compounds of theinvention do not include thalidomide.

[0049] Other specific immunomodulatory compounds of the inventioninclude, but are not limited to, 1-oxo-and 1,3dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines substituted with amino inthe benzo ring as described in U.S. Pat. No. 5,635,517 which isincorporated herein. These compounds have the structure I:

[0050] in which one of X and Y is C═O, the other of X and Y is C═O orCH₂, and R² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

[0051] 1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;

[0052] 1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;

[0053] 1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;

[0054] 1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;

[0055] 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;

[0056] and1,3-dioxo-2-(2,6-dioxopiperndin-3-yl)-5-aminoisoindoline.

[0057] Other specific immunomodulatory compounds of the invention belongto a class of substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as thosedescribed in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and6,476,052, and International Patent Application No. PCT/US97/13375(International Publication No. WO 98/03502), each of which isincorporated herein. Compounds representative of this class are of theformulas:

[0058] wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.optically pure (R) or (S) enantiomers) of these compounds.

[0059] Still other specific immunomodulatory compounds of the inventionbelong to a class of isoindole-imides disclosed in U.S. patentapplication Ser. Nos. 10/032,286 and 09/972,487, and InternationalApplication No. PCT/US01/50401(International Publication No. WO02/059106), each of which are incorporated herein by reference.Representative compounds are of formula II:

[0060] and pharmaceutically acceptable salts, hydrates, solvates,clathrates, enantiomers, diastereomers, racemates, and mixtures ofstereoisomers thereof, wherein:

[0061] one of X and Y is C═O and the other is CH₂ or C═O;

[0062] R¹ is H, (C₁-C₈ )alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl (C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

[0063] R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or(C₂-C₈)alkynyl;

[0064] R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alky-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

[0065] R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkyl-OR⁵, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

[0066] R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,or (C₂-C₅)heteroaryl;

[0067] each occurrence of R⁶ is independently H, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O-R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

[0068] n is 0 or 1; and

[0069] * represents a chiral-carbon center.

[0070] In specific compounds of formula II, when n is 0 then R1 is(C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl,(C0-C4)alkyl-(C 1-C6)heterocycloalkyl, (C₀-C4)alkyl-(C2-C5)heteroaryl,C(O)R3, C(O)OR4, (C₁-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5,(C1-C8)alkyl-C(O)OR5, C(S)NHR3, or (C1-C8)alkyl-O(CO)R5;

[0071] R² is H or (C₁-C₈)alkyl; and

[0072] R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂ ;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

[0073] In other specific compounds of formula II, R² is H or(C₁-C₄)alkyl.

[0074] In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl orbenzyl.

[0075] In other specific compounds of formula II, R¹ is H, (C₁-C8)alkyl,benzyl, CH₂OCH₃, CH₂CH₂OCH₃, or

[0076] In another embodiment of the compounds of formula II, R¹ is

[0077] wherein Q is O or S, and each occurrence of R⁷ is independentlyH, (C₁-C₈)alkyl, benzyl, CH₂OCH₃, or CH₂CH₂OCH₃.

[0078] In other specific compounds of formula II, R¹ is C(O)R³.

[0079] In other specific compounds of formula II, R³ is(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₁-C₈)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

[0080] In other specific compounds of formula II, heteroaryl is pyridyl,furyl, or thienyl.

[0081] In other specific compounds of formula II, R¹ is C(O)OR⁴.

[0082] In other specific compounds of formula II, the H of C(O)NHC(O)can be replaced with (C₁-C₄)alkyl, aryl, or benzyl.

[0083] Still other specific immunomodulatory compounds of the inventionbelong to a class of isoindole-imides disclosed in U.S. patentapplication Ser. No. 09/781,179, International Publication No. WO98/54170, and U.S. Pat. No. 6,395,754, each of which are incorporatedherein by reference. Representative compounds are of formula III:

[0084] and pharmaceutically acceptable salts, hydrates, solvates,clathrates, enantiomers, diastereomers, racemates, and mixtures ofstereoisomers thereof, wherein:

[0085] one of X and Y is C═O and the other is CH₂ or C═O;

[0086] R is H or CH2OCOR′;

[0087] (i) each of R¹, R², R³, or R⁴, independently of the others, ishalo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or(ii) one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹,R², R³, or R⁴ are hydrogen;

[0088] R⁵ is hydrogen or alkyl of 1 to 8 carbons

[0089] R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, orfluoro;

[0090] R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

[0091] R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which nhas a value of 0 to 4;

[0092] each of R8 and R9 taken independently of the other is hydrogen oralkyl of 1 to 8 carbon atoms, or R8 and R9 taken together aretetramethylene, pentamethylene, hexamethylene, or —CH₂CH₂[X]X₁CH₂CH₂— inwhich [X]X₁ is —O—, —S—, or —NH—;

[0093] R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

[0094] * represents a chiral-carbon center.

[0095] The most preferred immunomodulatory compounds of the inventionare 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (ACTIMID™)has the following chemical structure:

[0096] 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(REVIMID™) has the following chemical structure:

[0097] The compounds of the invention can either be commerciallypurchased or prepared according to the methods described in the patentsor patent publications disclosed herein. Further, optically purecompounds can be asymmetrically synthesized or resolved using knownresolving agents or chiral columns as well as other standard syntheticorganic chemistry techniques.

[0098] As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

[0099] Compounds that are acidic in nature are capable of forming saltswith various pharmaceutically acceptable bases. The bases that can beused to prepare pharmaceutically acceptable base addition salts of suchacidic compounds are those that form non-toxic base addition salts,i.e., salts containing pharmacologically acceptable cations such as, butnot limited to, alkali metal or alkaline earth metal salts and thecalcium, magnesium, sodium or potassium salts in particular. Suitableorganic bases include, but are not limited to,N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumaine (N-methylglucamine), lysine, and procaine.

[0100] As used herein and unless otherwise indicated, the term “prodrug”means a derivative of a compound that can hydrolyze, oxidize, orotherwise react under biological conditions (in vitro or in vivo) toprovide the compound. Examples of prodrugs include, but are not limitedto, derivatives of immunomodulatory compounds of the invention thatcomprise biohydrolyzable moieties such as biohydrolyzable amides,biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzablecarbonates, biohydrolyzable ureides, and biohydrolyzable phosphateanalogues. Other examples of prodrugs include derivatives ofimmunomodulatory compounds of the invention that comprise —NO, —NO₂,—ONO, or —ONO₂ moieties. Prodrugs can typically be prepared usingwell-known methods, such as those described in 1 Burger's MedicinalChemistry and Drug Discovery, 172-178, 949-982 (Manfred E. Wolff ed.,5th ed. 1995), and Design of Prodrugs (H. Bundgaard ed., Elselvier, NewYork 1985).

[0101] As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

[0102] It should be noted that if there is a discrepancy between adepicted structure and a name given that structure, the depictedstructure is to be accorded more weight. In addition, if thestereochemistry of a structure or a portion of a structure is notindicated with, for example, bold or dashed lines, the structure orportion of the structure is to be interpreted as encompassing allstereoisomers of it.

[0103] 4.2. Second Active Agents

[0104] One or more second active ingredients can be used in the methodsand compositions of the invention together with an immunomodulatorycompound of the invention. in a preferred embodiment, the second activeagents are capable of affecting or improving the process of blood cellproduction. Specific second active agents also stimulate the divisionand differentiation of committed erythroid progenitors in cells in vitroor in vivo.

[0105] Second active agents can be large molecules (e.g., proteins) orsmall molecules (e.g., synthetic inorganic, organometallic, or organicmolecules). The second active agents include but are not limited tohematopoietic growth factors, cytokines, anti-cancer agents,antibiotics, proteasome inhibitors, immunosuppressive agents and othertherapeutics discussed herein. Particular agents include, but are notlimited to, G-CSF, GM-CSF, EPO, dexamethasone, topotecan,pentoxifylline, irinotecan, ciprofloxacin, vinorelbine, IL2, IL8, IL18,Ara-C, isotretinoin, 13-cis-retinoic acid,12-O-tetradecanoylphorbol-13-acetate (TPA), 5-AZA2′-deoyxcytidine,9-nitrocamp-tothecin, transretinoic acid, amifostine, amphotericin B andliposomal amphotericin B, anti-CD-20 monoclonal antibody, anti-thymocyleglobulin (ATG), arsenic trioxide, azacytidine, bevacizumab, bismuthmonoclonal antibody, bryostatin, busulfan, caspofungin acetate,celocoxib, cladribine, cyclophosphamide, cyclosporine, cytarabine,cytosine, daunorubicin, depsipeptide, etoposide, farresy transferaseinhibitor, flavopiridol, Flt3 ligand, fludarabine, gentuzumab ozogomicin(mylotarg), etanercept (Enbrel®), imatinib (Glivec®), anti-TNF-αantibodies, infliximab (Remicade®), humanized monoclonal anti-VEGFantibody, idarubicine, leucovorin, melphalan, mitoxantrone, monoclonalantibody ABX-CBL, monoclonal antibody CD52, mycophenolate mofetil,oblimersen, omega-3 fatty acids, pentostatin, phenylbutyrate, PR1leukemia peptide vaccine, montanide, proteasome inhibitor, sodiumphenyl-butyrate, sodium salicylate, temozolomide, thymoglobulin,troxatyl, tumor necrosis factor receptor IgG chimera, Yttrium Y 90humanized monoclonal antibody M195. In a specific embodiment of theinvention, an immunomodulatory compound of the invention is used incombination with pentoxifylline, ciprofloxacin, and/or dexamethasone.

[0106] This invention also encompasses the use of native, naturallyoccurring, and recombinant proteins. The invention further encompassesmutants and derivatives (e.g., modified forms) of naturally occurringproteins that exhibit, in vivo, at least some of the pharmacologicalactivity of the proteins upon which they are based. Examples of mutantsinclude, but are not limited to, proteins that have one or more aminoacid residues that differ from the corresponding residues in thenaturally occurring forms of the proteins. Also encompassed by the term“mutants” are proteins that lack carbohydrate moieties normally presentin their naturally occurring forms (e.g., nonglycosylated forms).Examples of derivatives include, but are not limited to, pegylatedderivatives and fusion proteins, such as proteins formed by fusing IgG1or IgG3 to the protein or active portion of the protein of interest.See, e.g., Penichet, M. L. and Morrison, S. L., J. Immunol. Methods248:91-101 (2001).

[0107] Recombinant and mutated forms of G-CSF can be prepared asdescribed in U.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and5,580,755; all of which are incorporated herein by reference.Recombinant and mutated forms of GM-CSF can be prepared as described inU.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; all of which areincorporated herein by reference. In fact, recombinant forms of G-CSFand GM-CSF are currently sold in the United States for the treatment ofsymptoms associated with specific chemotherapies. A recombinant form ofG-CSF known as filgrastim is sold in the United States under the tradename NEUPOGEN®. NEUPOGEN® is known to stimulate division and maturationof granulocytes, mostly neutrophils, in MDS patients and to enhanceerythroid response in combination with EPO. Physicians' Desk Reference,587-592 (56^(th) ed., 2002). A recombinant form of GM-CSF known assargramostim is also sold in the United States under the trade nameLEUKINE®. LEUKINE® is known to stimulate division and maturation ofearlier myeloid and macrophage precursor cells and has been reported toincrease granulocytes. Physicians' Desk Reference, 1755-1760 (56^(th)ed., 2002). A recombinant form of EPO known as epoetin alfa is sold inthe United States under the trade name EPOGEN®. EPOGEN® is used tostimulate red cell production by stimulating division and maturation ofcommitted red cell precursor cells. EPOGEN® has been reported to beeffective in 20-26% of MDS patient when administered by itself and in asmany as 48% of patients when combined with G-CSF or GM-CSF. Physicians'Desk Reference, 582-587 (56^(th) ed., 2002).

[0108] A growth-factor or cytokine such as G-CSF, GM-CSF and EPO canalso be administered in the form of a vaccine. For example, vaccinesthat secrete, or cause the secretion of, cytokines such as G-CSF andGM-CSF can be used in the methods, pharmaceutical compositions, and kitsof the invention. See, e.g., Emens, L. A., et al., Curr. Opinion Mol.Ther. 3(1):77-84 (2001).

[0109] Other compounds that can be administered or used in combinationwith an immunomodulatory compound of the invention include thosedisclosed in U.S. provisional patent application No. 60/380,842, filedMay 17, 2002, and U.S. provisional patent application No. 60/380,843,filed May 17, 2002, both of which are incorporated herein by reference.

[0110] 4.3. Methods of Treatment and Management

[0111] Methods of this invention encompass methods of preventing,treating and/or managing various types of MDS. As used herein, unlessotherwise specified, the term “preventing” includes but is not limitedto, inhibition or the averting of symptoms associated with MDS. Thesymptoms associated with MDS include, but are not limited to, anemia,thrombocytopenia, neutropenia, cytopenia, bicytopenia (two deficientcell lines), and pancytopenia (three deficient cell lines). As usedherein, unless otherwise specified, the term “treating” refers to theadministration of a composition after the onset of symptoms of MDS,whereas “preventing” refers to the administration prior to the onset ofsymptoms, particularly to patients at risk of MDS. As used herein andunless otherwise indicated, the term “managing” encompasses preventingthe recurrence of MDS in a patient who had suffered from MDS,lengthening the time a patient who had suffered from MDS remains inremission, and/or preventing the occurrence of MDS in patients at riskof suffering from MDS.

[0112] The invention encompasses methods of treating or preventingpatients with primary and secondary MDS. It further encompasses methodstreating patients who have been previously treated for MDS, as well asthose who have not previously been treated for MDS. Because patientswith MDS have heterogenous clinical manifestations and varying clinicaloutcomes, it has become apparent that staging the patients according totheir prognosis and approaching therapy depending on the severity andstage is necessary. Indeed, the methods and compositions of thisinvention can be used in various stages of treatments for patients withone or more types of MDS including, but not limited to, refractoryanemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts(RAEB), RAEB in transformation (RAEB-T), or chronic myelomonocyticleukemia (CMML). The invention also contemplates treating patientsdiagnosed using the IPSS for MDS discussed above. Greenberg et al.,Blood 1997 (89):2079-88.

[0113] Methods encompassed by this invention comprise administering animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof to a patient (e.g., a human) suffering, or likely to suffer,from MDS. Specific patient populations include the elderly, i.e., ages60 and above as well as those over 35 years of age. Patients withfamilial history of MDS or leukemia are also preferred candidates forpreventive regimens.

[0114] In one embodiment of the invention, an immunomodulatory compoundof the invention is administered orally and in a single or divided dailydoses in an amount of from about 0.10 to about 150 mg/day. In aparticular embodiment,4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™) isadministered in an amount of from about 0.1 to about 1 mg per day, oralternatively about 5 mg every other day.3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) can be preferably administered in an amount of from about 5to 25 mg per day, or alternatively from about 25 to about 50 mg everyother day.

[0115] 4.3.1 Combination Therapy With A Second Active Agent

[0116] Particular methods of the invention comprise comprisesadministering 1) an immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof, and 2) a second active agent or activeingredient. Examples of immunomodulatory compounds of the invention aredisclosed herein (see, e.g., section 4.1); and examples of the secondactive agents are also disclosed herein (see, e.g., section 4.2).

[0117] Administration of the immunomodulatory compounds and the secondactive agents to a patient can occur simultaneously or sequentially bythe same or different routes of administration. The suitability of aparticular route of administration employed for a particular activeagent will depend on the active agent itself (e.g., whether it can beadministered orally without decomposing prior to entering the bloodstream) and the disease being treated. A preferred route ofadministration for an immunomodulatory compound is oral. Preferredroutes of administration for the second active agents or ingredients ofthe invention are known to those of ordinary skill in the art. See,e.g., Physicians' Desk Reference, 1755-1760 (56^(th) ed., 2002).

[0118] In one embodiment, the second active agent is administeredintravenously or subcutaneously and once or twice daily in an amount offrom about 1 to about 1000 mg, from about 5 to about 500 mg, from about10 to about 350 mg, or from about 50 to about 200 mg. The specificamount of the second active agent will depend on the specific agentused, the type of MDS being treated or managed, the severity and stageof MDS, and the amount(s) of immunomodulatory compounds of the inventionand any optional additional active agents concurrently administered tothe patient. In a particular embodiment, the second active agent isGM-CSF, G-CSF, EPO, transretinoic acid, dexamethasone, topotecan,pentoxifylline, ciprofloxacin, dexamethasone, IL2, IL8, IL18, Ara-C,vinorelbine, or a combination thereof. GM-CSF is administered in anamount of from about 60 to about 500 mcg/m² intravenously over 2 hours,or from about 5 to about 12 mcg/m²/day subcutaneously. G-CSF isadministered subcutaneously in an amount of about 1 mcg/kg/day initiallyand can be adjusted depending on rise of total granulocyte counts. Themaintenance dose is 300 (in smaller patients) or 480 mcg subcutaneously.EPO is administered subcutaneously in an amount of 10,000 Unit 3 timesper week.

[0119] 4.3.2 Use With Transplantation Therapy

[0120] In still another embodiment, this invention encompasses a methodof treating, preventing and/or managing MDS, which comprisesadministering the immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof, in conjunction with transplantationtherapy. As discussed elsewhere herein, the treatment of MDS is based onthe stages and mechanism of the disease. As inevitable leukemictransformation develops in certain stages of MDS, transplantation ofperipheral blood stem cells, hematopoietic stem cell preparation or bonemarrow may be necessary. The combined use of the immunomodulatorycompound of the invention and transplantation therapy provides a uniqueand unexpected synergism. In particular, an immunomodulatory compound ofthe invention exhibits immunomodulatory activity that may provideadditive or synergistic effects when given concurrently withtransplantation therapy in patients with MDS. An immunomodulatorycompound of the invention can work in combination with transplantationtherapy reducing complications associated with the invasive procedure oftransplantation and risk of related Graft Versus Host Disease (GVHD).This invention encompasses a method of treating, preventing and/ormanaging MDS which comprises administering to a patient (e.g., a human)an immunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, before, during, or after the transplantation of umbilical cordblood, placental blood, peripheral blood stem cell, hematopoictic stemcell preparation or bone marrow. Examples of stem cells suitable for usein the methods of the invention are disclosed in U.S. provisional patentapplication No. 60/372,348, filed Apr. 12, 2002 by R. Hariri et al., theentirety of which is incorporated herein by reference.

[0121] 4.3.3. Cycling Therapy

[0122] In certain embodiments, the prophylactic or therapeutic agents ofthe invention are cyclically administered to a patient. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of the agent and/or the second agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

[0123] In a particular embodiment, prophylactic or therapeutic agentsare administered in a cycle of about 16 weeks, about once or twice everyday. One cycle can comprise the administration of a therapeutic orprophylactic agent and at least one (1) or three (3) weeks of rest. Thenumber of cycles administered is from about 1 to about 12 cycles, moretypically from about 2 to about 10 cycles, and more typically from about2 to about 8 cycles.

[0124] 4.4. Pharmaceutical Compositions and Single Unit Dosage Forms

[0125] Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms of the invention comprise an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof. Pharmaceutical compositionsand dosage forms of the invention can further comprise one or moreexcipients.

[0126] Pharmaceutical compositions and dosage forms of the invention canalso comprise one or more additional active ingredients. Consequently,pharmaceutical compositions and dosage forms of the invention comprisethe active ingredients disclosed herein (e.g., an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and asecond active ingredient). Examples of optional additional activeingredients are disclosed herein (see, e.g., section 4.2).

[0127] Single unit dosage forms of the invention are suitable for oral,mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), orparenteral (e.g., subcutaneous, intravenous, bolus injection,intramuscular, or intraarterial), transdermal or transcutaneousadministration to a patent. Examples of dosage forms include, but arenot limited to: tablets; caplets; capsules, such as soft elastic gelatincapsules; cachets; troches; lozenges; dispersions; suppositories;powders; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a patient,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

[0128] The composition, shape, and type of dosage forms of the inventionwill typically vary depending on their use. For example, a dosage formused in the acute treatment of a disease may contain larger amounts ofone or more of the active ingredients it comprises than a dosage formused in the chronic treatment of the same disease. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease. These and other ways in which specific dosage formsencompassed by this invention will vary from one another will be readilyapparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

[0129] Typical pharmaceutical compositions and dosage forms comprise oneor more excipients. Suitable excipients are well known to those skilledin the art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition. Consequently, this invention encompasses pharmaceuticalcompositions and dosage forms that contain little, if any, lactose othermono- or di-saccharides. As used herein, the term “lactose-free” meansthat the amount of lactose present, if any, is insufficient tosubstantially increase the degradation rate of an active ingredient.

[0130] Lactose-free compositions of the invention can compriseexcipients that are well known in the art and are listed, for example,in the U.S. Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-freecompositions comprise active ingredients, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Preferred lactose-free dosage forms comprise activeingredients, microcrystalline cellulose, pre-gelatinized starch, andmagnesium stearate.

[0131] This invention further encompasses anhydrous pharmaceuticalcompositions and dosage forms comprising active ingredients, since watercan facilitate the degradation of some compounds. For example, theaddition of water (e.g., 5%) is widely accepted in the pharmaceuticalarts as a means of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed.. Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

[0132] Anhydrous pharmaceutical compositions and dosage forms of theinvention can be prepared using anhydrous or low moisture containingingredients and low moisture or low humidity conditions. Pharmaceuticalcompositions and dosage forms that comprise lactose and at least oneactive ingredient that comprises a primary or secondary amine arepreferably anhydrous if substantial contact with moisture and/orhumidity during manufacturing, packaging, and/or storage is expected.

[0133] An anhydrous pharmaceutical composition should be prepared andstored such that its anhydrous nature is maintained. Accordingly,anhydrous compositions are preferably packaged using materials known toprevent exposure to water such that they can be included in suitableformulary kits. Examples of suitable packaging include, but are notlimited to, hermetically sealed foils, plastics, unit dose containers(e.g., vials), blister packs, and strip packs.

[0134] The invention further encompasses pharmaceutical compositions anddosage forms that comprise one or more compounds that reduce the rate bywhich an active ingredient will decompose. Such compounds, which arereferred to herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0135] Like the amounts and types of excipients, the amounts andspecific types of active ingredients in a dosage form may differdepending on factors such as, but not limited to, the route by which itis to be administered to patients. However, typical dosage forms of theinvention comprise an immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof in an amount of from about 0.10 to about150 mg. Typical dosage forms comprise an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof in an amount of about 0.1,1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150 or 200 mg. In aparticular embodiment, a preferred dosage form comprises4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™) inan amount of about 1, 2, 5, 10, 25 or 50 mg. In a specific embodiment, apreferred dosage form comprises3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) in an amount of about 5, 10, 25 or 50 mg. Typical dosageforms comprise the second active ingredient in an amount of 1 to about1000 mg, from about 5 to about 500 mg, from about 10 to about 350 mg, orfrom about 50 to about 200 mg. Of course, the specific amount of thesecond active ingredient will depend on the specific agent used, thetype of MDS being treated or managed, and the amount(s) ofimmunomodulatory compounds of the invention, and any optional additionalactive agents concurrently administered to the patient.

[0136] 4.4.1 Oral Dosage Forms

[0137] Pharmaceutical compositions of the invention that are suitablefor oral administration can be presented as discrete dosage forms, suchas, but are not limited to, tablets (e.g., chewable tablets), caplets,capsules, and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

[0138] Typical oral dosage forms of the invention are prepared bycombining the active ingredients in an intimate admixture with at leastone excipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

[0139] Because of their ease of administration, tablets and capsulesrepresent the most advantageous oral dosage unit forms, in which casesolid excipients are employed. If desired, tablets can be coated bystandard aqueous or nonaqueous techniques. Such dosage forms can beprepared by any of the methods of pharmacy. In general, pharmaceuticalcompositions and dosage forms are prepared by uniformly and intimatelyadmixing the active ingredients with liquid carriers, finely dividedsolid carriers, or both, and then shaping the product into the desiredpresentation if necessary.

[0140] For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

[0141] Examples of excipients that can be used in oral dosage forms ofthe invention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

[0142] Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, PA), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

[0143] Examples of fillers suitable for use in the pharmaceuticalcompositions and dosage forms disclosed herein include, but are notlimited to, talc, calcium carbonate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, andmixtures thereof. The binder or filler in pharmaceutical compositions ofthe invention is typically present in from about 50 to about 99 weightpercent of the pharmaceutical composition or dosage form.

[0144] Disintegrants are used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Tablets that contain too much disintegrant may disintegratein storage, while those that contain too little may not disintegrate ata desired rate or under the desired conditions. Thus, a sufficientamount of disintegrant that is neither too much nor too little todetrimentally alter the release of the active ingredients should be usedto form solid oral dosage forms of the invention. The amount ofdisintegrant used varies based upon the type of formulation, and isreadily discernible to those of ordinary skill in the art. Typicalpharmaceutical compositions comprise from about 0.5 to about 15 weightpercent of disintegrant, preferably from about 1 to about 5 weightpercent of disintegrant.

[0145] Disintegrants that can be used in pharmaceutical compositions anddosage forms of the invention include, but are not limited to,agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

[0146] Lubricants that can be used in pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

[0147] A preferred solid oral dosage form of the invention comprises animmunomodulatory compound of the invention, anhydrous lactose,microcrystalline cellulose, polyvinylpyrrolidone, stearic acid,colloidal anhydrous silica, and gelatin.

[0148] 4.4.2 Delayed Release Dosage Forms

[0149] Active ingredients of the invention can be administered bycontrolled release means or by delivery devices that are well known tothose of ordinary skill in the art. Examples include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767,5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of whichis incorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

[0150] All controlled-release pharmaceutical products have a common goalof improving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

[0151] Most controlled-release formulations are designed to initiallyrelease an amount of drug (active ingredient) that promptly produces thedesired therapeutic effect, and gradually and continually release ofother amounts of drug to maintain this level of therapeutic orprophylactic effect over an extended period of time. In order tomaintain this constant level of drug in the body, the drug must bereleased from the dosage form at a rate that will replace the amount ofdrug being metabolized and excreted from the body. Controlled-release ofan active ingredient can be stimulated by various conditions including,but not limited to, pH, temperature, enzymes, water, or otherphysiological conditions or compounds.

[0152] 4.4.3 Parenteral Dosage Forms

[0153] Parenteral dosage forms can be administered to patients byvarious routes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

[0154] Suitable vehicles that can be used to provide parenteral dosageforms of the invention are well known to those skilled in the art.Examples include, but are not limited to: Water for Injection USP;aqueous vehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

[0155] Compounds that increase the solubility of one or more of theactive ingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention. For example, cyclodextrin andits derivatives can be used to increase the solubility of animmunomodulatory compound of the invention, and its derivatives. See,e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

[0156] 4.4.4 Topical and Mucosal Dosage Forms

[0157] Topical and mucosal dosage forms of the invention include, butare not limited to, sprays, aerosols, solutions, emulsions, suspensions,or other forms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th) and 18^(th) eds., Mack Publishing,Easton Pa. (1980 & 1990); and Introduction to Pharmaceutical DosageForms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage formssuitable for treating mucosal tissues within the oral cavity can beformulated as mouthwashes or as oral gels.

[0158] Suitable excipients (e.g., carriers and diluents) and othermaterials that can be used to provide topical and mucosal dosage formsencompassed by this invention are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form solutions, emulsions or gels, which arenon-toxic and pharmaceutically acceptable. Moisturizers or humectantscan also be added to pharmaceutical compositions and dosage forms ifdesired. Examples of such additional ingredients are well known in theart. See, e.g., Remington 's Pharmaceutical Sciences, 16^(th) and18^(th) eds., Mack Publishing, Easton Pa. (1980 & 1990).

[0159] The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

[0160] 4.4.5 Kits

[0161] Typically, active ingredients of the invention are preferably notadministered to a patient at the same time or by the same route ofadministration. This invention therefore encompasses kits which, whenused by the medical practitioner, can simplify the administration ofappropriate amounts of active ingredients to a patient.

[0162] A typical kit of the invention comprises a dosage form of animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt salt, solvate, hydrate, stereoisomer, prodrug, orclathrate thereof. Kits encompassed by this invention can furthercomprise additional active ingredients such as G-CSF, GM-CSF, EPO,topotecan, pentoxifylline, ciprofloxacin, dexamethasone, IL2, IL8, IL18,Ara-C, vinorelbine, isotretinoin, 13-cis-retinoic acid, or apharmacologically active mutant or derivative thereof, or a combinationthereof. Examples of the additional active ingredients include, but arenot limited to, those disclosed herein (see, e.g., section 4.2).

[0163] Kits of the invention can further comprise devices that are usedto administer the active ingredients. Examples of such devices include,but are not limited to, syringes, drip bags, patches, and inhalers.

[0164] Kits of the invention can further comprise cells or blood fortransplantation as well as pharmaceutically acceptable vehicles that canbe used to administer one or more active ingredients. For example, if anactive ingredient is provided in a solid form that must be reconstitutedfor parenteral administration, the kit can comprise a sealed containerof a suitable vehicle in which the active ingredient can be dissolved toform a particulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5. EXAMPLES

[0165] The following studies are intended to further illustrate theinvention without limiting its scope.

[0166] Excessive production of the growth inhibitory cytokine TNF-α isdemonstrated in bone marrow plasma of patients with MDS, implicatingTNF-α as a critical negative regulator of erythroid progenitor survivalin the disorder. As a result, a study with an immunomodulatory compoundof the invention was conducted.

[0167] 5.1. Pharmacology and Toxicology Studies

[0168] A series of non-clinical pharmacology and toxicology studies havebeen performed to support the clinical evaluation of an immunomodulatorycompound of the invention in human subjects. These studies wereperformed in accordance with internationally recognized guidelines forstudy design and in compliance with the requirements of Good LaboratoryPractice (GLP), unless otherwise noted.

[0169] The pharmacological properties of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione,including activity comparisons with thalidomide, have been characterizedin in vitro studies. Studies examined the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orthalidomide on the production of various cytokines. In all studies,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione was atleast 50 times more potent than thalidomide. In addition, a safetypharmacology study of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione hasbeen conducted in dogs and the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on ECGparameters were examined further as part of three repeat-dose toxicitystudies in primates. The results of these studies are described below.

[0170] 5.2. Modulation of Cytokine Production

[0171] Inhibition of TNF-α production following LPS-stimulation of humanPBMC and human whole blood by3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orthalidomide was investigated in vitro (Muller et al., Bioorg. Med. Chem.Lett. 9:1625-1630, 1999). The IC₅₀'s of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione forinhibiting production of TNF-α following LPS-stimulation of PBMC andhuman whole blood were ˜100 nM (25.9 ng/mL) and ˜480 nM (103.6 ng/mL),respectively. Thalidomide, in contrast, had an IC₅₀ of ˜194 μM (50.2μg/mL) for inhibiting production of TNF-α following LPS-stimulation ofPBMC.

[0172] n vitro studies suggest a pharmacological activity profile for3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione that issimilar to, but 50 to 2000 times more potent than, thalidomide. Thepharmacological effectsof3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionederive from its action as an inhibitor of cellular response toreceptor-initiated trophic signals (e.g., IGF-1, VEGF,cyclooxygenase-2), and other activities. As a result,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionesuppresses the generation of inflammatory cytokines, down-regulatesadhesion molecules and apoptosis inhibitory proteins (e.g., cFLIP,cIAP), promotes sensitivity to death-receptor initiated programmed celldeath, and suppresses angiogenic response. The studies show that3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dioneabrogates mitogenic response to VEGF in AML cells by extinguishingligant-induced Akt-phosphorylation, and selectively suppresses MDS vsnormal bone marrow proginitor formation in pre-clinical models.

[0173] 5.3. Clinical Studies in MDS Patients

[0174] Protocol

[0175] An immunomodulatory compound of the invention, such as4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, isadministered in an amount of from about 0.1 to about 25 mg per day topatients with MDS for 16 weeks, who are subsequently evaluated for ahematological response. Response rates are assessed in cohortsstratified by the likelihood of an MDS subtype to transform to leukemiaaccording to the International Prognostic Scoring System (IPSS)-definedrisk groups (i.e., IPSS Low and Intermediate I; versus IPSS IntermediateII and High).

[0176] For example, fifteen patients are enrolled in the first cohortand receive treatment with 25 mg per day of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thenumber of patients who subsequently experience an erythroid response(major or minor response) by week 16 is evaluated. If no responses areobserved, the study is terminated due to lack of efficacy. If, however,4 or more patients respond, the study is terminated due to promisingclinical activity. In the intermediate case (e.g., 1, 2 or 3 patientsrespond), a second cohort of 10 patients is enrolled. If after thecompletion of treatment by the second cohort, 4 or more patients respondamong the 25 patients treated, it is concluded that the3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione showspromising clinical activity.

[0177] Clinical Study

[0178] Clinical studies were performed for the remitting potential of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione in MDSpatients with red blood cell transfusion-dependence (>4 units/8 weeks)or symptomatic anemia (Hgb<10 g/dl). Patients received continuoustreatment with3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione at aoral dose of 25 mg daily. Responses were assessed according to IWGcriteria after 16 weeks of treatments. Among 15 patients receiving thetreatments, 11 patients were evaluable for toxicity, nine patients wereevaluable for response (>8 wks therapy), and three patients discontinuedthe therapy prematurely (<2 weeks) due to cholecystitis, autoimmunehemolytic anemia, or patient refusal. Median age of the patients was 78years ranging from 51 to 82 years. FAB types of the MDS patients includeRA [4 patients], RARS [4 patients], RAEB [6 patients], and RAEB-T[1patient] with corresponding IPSS categories of Low/Int-1 in 11patients and Int-2/High in four patients. Myelosuppression, which wascharacterized by higher than grade 3 common toxicity criteria or 50%decrease in leukocyte and platelet counts [9 patients], and grade 3fatigue [1 patient], necessitated dose reduction to 10 mg in the initialten patients. All subsequent patients initiated oral administrationswith 10 mg daily. Grade 1,2 drug-related adverse effects were limited tothe 25 mg dose and included pruritus or itchy scalp [6 patients] andmyalgia [1 patient]. Six (66%) of nine evaluable patients experiencedhematologic benefit (dual lineage, 1 patient), including {fraction(6/7)} (86%) patients with IPSS Low/Int-1. Hematologic responsesincluded RBC transfusion-independence [4 patients], decrease in RBCtransfusions of more than 50% [1 patient], increase in Hgb of more than1.5 g [1 patient], and one minor platelet response (increase of morethan 30,000//uL ). Among five patients evaluable for cytogeneticresponse, three patients achieved either a complete or partial (decreasein abnormal metaphases of more than 50%) remission. Responses wereassociated with normalization of blast percentage [1 patient], reducedgrade of BM cytologic dysplasia, and 50% to more than 40 timesimprovement in BM multipotent progenitor (CFU-GEMM) and erythroid burst(BFU-E) formation. Correlation with changes in apoptotic index,angiogenic features (cellular/plasma VEGF, microvessel density),cytokine generation, and proliferative fraction (Ki67) are in progress.The results of this study indicate that3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione hasremarkable erythropoietic and cytogenetic remitting activity in patientswith low/intermediate-1 risk MDS. Clinical benefit appears greatest inpatients with low/intermediate-1 disease or the 5q-syndrome, associatedwith resolution of cytology dysplasia. The increase in apoptotic index,restoration of CFC, and suppression of karyotypic abnormalities suggestthat the compound accelerates extinction of myelodysplastic clones.Based upon these data, the study has been expanded to treat additionalsubjects. Treatment with 10 mg as a continuous oral daily dose iswell-tolerated with minimal myelosuppression.

[0179] Expanded Study

[0180] The clinical study was expanded with additional 16 MDS patientsfor at least eight weeks. According to the IPSS, 13 of these patientswere categorized as low- or intermediate-1-risk patients and threepatients were grouped as intermediate-2- or high-risk patients.According to the FAB classification, there were 11 patients withrefractory anemia (RA) or RA with ringed sideroblasts (RARS), and fivepatients with RA with excess blasts (RAEB), RAEB in transformation(RAEB-T). The starting dose of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione was 25mg daily for the first 13 patients and 10 mg daily for the remainingthree patients. All patients receiving the starting dose of 25 mgrequired dose reduction by the completion of eight weeks therapy. Amongthese 16 patients who completed at least 8 weeks of monitoring, ninepatients achieved an erythroid response as assessed by the InternationalMDS Working Group Criteria. The erythroid responses consisted oftransfusion independence in seven previously transfusion-dependentpatients, a >2 g/dL rise in blood hemoglobin concentration in onepatient in with transfusion-independent anemia, and a >50 % decrease inRBC transfusion requirement in one transfusion-dependent patient.Therefore, a major erythroid response developed in eight of 16 patientsand a minor erythroid response was observed in one patient. All of ninepatients who showed erythroid response were low- or intermediate-1-riskpatients. One patient also had a minor platelet response. In addition,complete cytogenetic responses developed in five in eight patients withabnormal karyotypes at baseline. These five patients with completecytogenetic responses all had the Del5q31-33 abnormality, which has beendiscovered to be a good prognostic factor for MDS. Indeed, all fivepatients who enrolled in this study with 5q-syndrome achieved a completecytogenetic response and a major erythroid response. The study alsoindicated an association of this therapy with an increased apoptoticindex for myelodysplastic progenitors and recovery of normalhematopoietic progenitor cells.

[0181] 5.4. Cycling Therapy in MDS Patients

[0182] As mentioned above, immunomodulatory compounds of the inventioncan be cyclically administered to patients with MDS. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of the agent and/or the second agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

Example 1

[0183] In a specific embodiment, prophylactic or therapeutic agents areadministered in a cycle of about 16 weeks, about once or twice everyday. One cycle can comprise the administration of a therapeutic onprophylactic agent and at least one (1), two (2), or three (3) weeks ofrest. The number of cycles administered is from about 1 to about 12cycles, more typically from about 2 to about 10 cycles, and moretypically from about 2 to about 8 cycles.

Example 2

[0184] The objectives of the study are to evaluate the efficacy andsafety of oral administration of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione inpatients with MDS. Patients receive the compound in an amount of 10 mg/dor 15 mg/d for 21 days every 28 days in 4-week cycles for 16 weeks (4cycles) or 24 weeks (6 cycles). The subject population comprisespatients with low- or interemediate-1-risk MDS (International PrognosticScoring System) with red blood cell transfusion-dependent anemia whohave received at least two units of RBCs within 8 week of baseline(first day of study treatment). In addition to hematological laboratorymonitoring, bone marrow aspirates/biopsies with cytogenic analyses areobtained at baseline, after the completion of 3 cycles and after thecompletion of 6 cycles. The bone marrow, safety and efficacy data arereviewed to assess benefit-to-risk considerations throughout the study.The study reviews red blood cell transfusion independence and majorerythroid response according to the International MDS Working GroupCriteria. Further, the study observes red blood cell transfusionindependence in the subgroup of patients with the 5q deletioncytogenetic abnormality; platelet, neutrophil, bone marrow andcytogenetic responses; and minor erythroid response of >50 % but <100 %reduction in red blood cell transfusion requirement over an 8 weekperiod. The study further monitors adverse events, hematological tests,serum chemistries, TSH, urinalysis, urine or serum pregnancy tests,vital signs, ECG and physical examinations.

Example 3

[0185] The objectives of the study are to compare the efficacy andsafety of oral administration of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione to thatof placebo plus standard care in patients with MDS. Patients receive thetherapy in 4-week cycles for 16 weeks (4 cycles) or 24 weeks (6 cycles).The subject population comprise patients with low- orinteremediate-1-risk MDS (International Prognostic Scoring System) withred blood cell transfusion-dependent anemia who have received at leasttwo units of RBCs within 8 week of baseline (first day of studytreatment). The study visits to assess safety and efficacy occur every 4weeks and hematologic laboratory monitoring is performed every 2 weeks.Bone marrow aspirates/biopsies with cytogenetic analyses are obtained atbaseline after the completion of 3 cycles and after the completion of 6cycles. Bone marrow findings, safety and efficacy data are reviewed toassess benefit-to-risk considerations throughout the study. An extensionstudy of continued treatments with the administration of the compound isavailable for patients who derive clinical benefit from 6 cycles of thetherapy and to provide an opportunity for subjects who were randomizedto placebo to cross over to the therapy.

[0186] Embodiments of the invention described herein are only a samplingof the scope of the invention. The full scope of the invention is betterunderstood with reference to the attached claims.

1-38. (canceled)
 39. A method of treating a myelodysplastic syndrome,which comprises administering to a patient in need thereof atherapeutically effective amount of a compound of formula (I):

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof,wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl.
 40. The method of claim 39, wherein R ishydrogen.
 41. The method of claim 39, wherein the compound has formula(I):

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl.
 42. The method of claim 39, wherein thecompound is a pharmaceutically acceptable salt.
 43. The method of claim39, wherein the compound is a pharmaceutically acceptable solvate. 44.The method of claim 39, wherein the compound is a pharmaceuticallyacceptable stereoisomer.
 45. The method of claim 44, wherein thestereoisomer is an enantiomerically pure R isomer.
 46. The method ofclaim 44, wherein the stereoisomer is an enantiomerically pure S isomer.47. The method of claim 39, wherein the compound is4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
 48. Themethod of claim 39, wherein the compound is3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. 49.The method of claim 39, which further comprises administering atherapeutically or prophylactically effective amount of a second activeagent.
 50. The method of claim 49, wherein the second active agent iscapable of improving blood cell production.
 51. The method of claim 49,wherein the second active agent is a cytokine, hematopdietic growthfactor, an anti-cancer agent, an antibiotic, a proteasome inhibitor, oran immunosuppressive agent.
 52. The method of claim 51, wherein thesecond active agent is etanercept, imatinib, anti-TNF-α antibodies,infliximab, G-CSF, GM-CSF, EPO, topotecan, pentoxifylline,ciprofloxacin, irinotecan, vinblastine, dexamethasone, IL2, IL8, IL18,Ara-C, vinorelbine, isotretinoin, 13-cis-retinoic acid, arsenic trioxideor a pharmacologically active mutant or derivative thereof.
 53. Themethod of claim 39, wherein the myelodysplastic syndrome is refractoryanemia, refractory anemia with ringed sideroblasts, refractory anemiawith excess blasts, refractory anemia with excess blasts intransformation, or chronic myelomonocytic leukemia.
 54. The method ofclaim 39, wherein the compound or a pharmaceutically acceptable salt,solvate or stereoisomer thereof is administered before, during or aftertransplanting umbilical cord blood, placental blood, peripheral bloodstem cell, hematopoietic stem cell preparation or bone marrow in thepatient.